ChromatographyChromatography is a popular and widely used method of purification. Chromatography is based on the differences in the adsorption and the solubility of the compounds to be separated. It involves two phases - a stationary phase and a moving (mobile) phase. The moving phase actually carries the compounds along the stationary phase. Depending on the interactions between the compounds and the stationary phase, the compounds get held up or slowed down by the stationary phase.
Thin-layer Chromatography (TLC)
Thin-layer chromatography is used for the separation of small quantities of the desired compound, especially for qualitative analysis. It is a widely used technique. In TLC, the liquid (mobile) phase is allowed to run along a layer of adsorbent. This thin layer of adsorbent (usually alumina or silica gel) is usually coated on a plate. After the sample (mixture dissolved in a solvent) is put onto the plate (usually a spot of sample is placed at the bottom of the plate), the plate is held vertical with a small amount of the solvent just touching the bottom of the plate. Due to capillary action, the solvent will ascend up the plate.
The sample will be separated between the various phases. The separation occurs as a result of differences in the rate of mobilities of the individual components of the sample. This is because of the difference in the affinities of the solute with respect to the two phases. As a rule of thumb, the higher the polarity of the compound (component) is, the lesser the rate of upward mobility of that compound. This generalization is based on the fact that the stationary phase used is highly polar. Polar substances bind strongly to the stationary phase. Also, the moving phase generally used is less polar with respect to the adsorbent. The separated and migrated components will appear as a series of spots on the plate. If the components are colorless, certain reagents are used to make the spots visible. Choosing the correct solvent is extremely important, and is best achieved by experience. Sometimes the best choice of solvent can be attained only by trial and error.
Rf value is the ratio of the distance traveled by a compound to the distance traveled by the solvent.
Using known Rf values of compounds under the same conditions, we can make sure that the desired compound is present in the mixture and can be separated. Rf value is a constant (under specific conditions) for a given compound, and if the TLC is done exactly under the same conditions specified, we can be reasonably sure about the identity of the compound. Keep in mind that it is always safe to make sure of the identity of a compound with other qualitative analysis as well.
In addition to the function of identifying compounds, TLC is also used to monitor the progression of reactions, and to test the authenticity or the effectiveness of other separation techniques.
Gas-liquid Chromatography (GLC)
As the name implies, a gas phase (moving phase) and a liquid phase (stationary phase) are involved in gas-liquid chromatography. Gas-liquid chromatography is usually employed for the separation of volatile compounds. The device used for this is called a gas chromatograph. Usually a syringe is used to inject the sample into the device, and the sample injected is readily turned into vapor form. This vaporized form of the sample is carried along the flow of an inert gas. The sample is passed through a column containing the adsorbent (liquid-stationary phase). The column is the site where the separation of the components occurs. The separated samples as they emerge from the column are detected using a detection device.
The maintenance of the correct temperature of the column is extremely important for an efficient separation of the components. The choice of the stationary liquid phase is also important, and so is the maintenance of an optimum rate of flow of the sample through the column. The latter can be maintained by controlling the flow of the carrier gas. This technique is highly sensitive. Hence, it can be used to detect compounds even if very little of the compound is present (as low as in the order of micrograms).
Technological developments in the field of identification of compounds using infra red and NMR techniques have revolutionized the ease with which we can analyze and identify even the most complex structures in organic chemistry. For the MCAT, we should be familiar with the techniques and the basic principles that are used in this field.